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QC Report

Report QC介紹基本信息，說(shuō)明：

• 報(bào)告發(fā)布時(shí)間

• 報(bào)告類(lèi)型atac

• 比對(duì)基因組使用的是什么，如mm10,hg38,rn6

• 比對(duì)軟件bowtie2

• 測(cè)序類(lèi)型為雙端測(cè)序（paired end）

• peak caller 軟件為 macs2

Alignment quality metrics (比對(duì)質(zhì)量檢測(cè))

SAMstat (raw unfiltered BAM) (原始比對(duì)質(zhì)量檢測(cè))

原始數(shù)據(jù)比對(duì)基因組過(guò)程的質(zhì)控：

• Total Reads，為全部reads數(shù)。

• duplicate Reads，為重復(fù)reads數(shù)，需要去除。

• Mapped Reads，為mapping到基因組上的reads數(shù)。

• % Mapped Reads，為reads的mapping率。
  （大于90%非常好，大于80%較好，較低時(shí)可考慮是否是實(shí)驗(yàn)的特殊處理或其他污染導(dǎo)致。）

• Properly Paired Reads，正確匹配的雙端reads。

• % Properly Paired Reads，reads 雙端匹配率。

• With itself，雙端匹配到基因組上的reads。

• singleton，只有單端匹配到基因組上的reads。

• % singleton，只有單端的匹配率。

• Diff.Chroms，沒(méi)有匹配到基因組上的數(shù)據(jù)。

Marking duplicates (filtered BAM) (重復(fù)數(shù)據(jù)標(biāo)記檢測(cè))

Filtered out (samtools view -F 1804):

• read unmapped (0x4)

• mate unmapped (0x8, for paired-end)

• not primary alignment (0x100)

• read fails platform/vendor quality checks (0x200)

• read is PCR or optical duplicate (0x400)

說(shuō)明：
Duplicate Reads是由于PCR過(guò)度擴(kuò)增，導(dǎo)致的完全一致的reads，這些reads在分析時(shí)需要去除。利用samtools過(guò)濾掉的數(shù)據(jù)包括：

• unmapped的reads（單端/雙端）

• 沒(méi)有唯一匹配的reads

• 本身質(zhì)量差的reads

• PCR重復(fù)產(chǎn)生的duplicate reads

SAMstat (filtered/deduped BAM) (過(guò)濾掉線粒體reads及重復(fù)reads后的質(zhì)量檢測(cè))

Filtered and duplicates removed

說(shuō)明：

• duplicate reads，對(duì)過(guò)濾后的reads進(jìn)行質(zhì)控。表格說(shuō)明同上。

Sequence quality metrics (filtered/deduped BAM) (測(cè)序質(zhì)量檢測(cè))

Open chromatin assays are known to have significant GC bias. Please take this            into consideration as necessary.

說(shuō)明：

• 紅色線條為每條序列在基因組特定位置的均一化覆蓋度，

• 藍(lán)色線條為每條序列的平均堿基的質(zhì)量

• 綠色線條為每條堿基的GC含量，正常一般分布在50%左右，

每條序列的平均堿基的質(zhì)量參考說(shuō)明：

Phred分值	不正確的堿基識(shí)別	堿基正確識(shí)別率	Q.sorce
10	1/10	90%	Q10
20	1/100	99%	Q20
30	1/1000	99.9%	Q30
40	1/10000	99.99%	Q40

Library complexity quality metrics (文庫(kù)復(fù)雜度質(zhì)量檢測(cè))

Library complexity (filtered non-mito BAM) (文庫(kù)復(fù)雜度)

Mitochondrial reads are filtered out by default.        The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads        in a dataset; it is the ratio between the number of positions in the genome        that uniquely mapped reads map to and the total number of uniquely mappable        reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations        with EXACTLY one read pair over the genomic locations with AT LEAST one read        pair. PBC1 is the primary measure, and the PBC1 should be close to 1.        Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking,        0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is        the ratio of genomic locations with EXACTLY one read pair over the genomic        locations with EXACTLY two read pairs. The PBC2 should be significantly        greater than 1. See more details at                the ENCODE portal standard for ChIP-Seq pipeline

NRF (non redundant fraction)
       PBC1 (PCR Bottleneck coefficient 1)
       PBC2 (PCR Bottleneck coefficient 2)
       PBC1 is the primary measure. Provisionally

• 0-0.5 is severe bottlenecking

• 0.5-0.8 is moderate bottlenecking

• 0.8-0.9 is mild bottlenecking

• 0.9-1.0 is no bottlenecking

說(shuō)明：

• Total Fragments:總片段數(shù)

• Distinct Fragments:可以唯一匹配到基因組上的片段數(shù)

• One Reads：只有一個(gè)reads匹配到特定位置的片段數(shù)

• Two Read：有兩個(gè)reads匹配的同一位置的片段數(shù)

• NRF = Distinct/Total

• PBC1 = OneRead/Distinct

• PBC2 = OneRead/TwoRead

文庫(kù)復(fù)雜度的指標(biāo)由三個(gè)值決定，NRF、PBC1、PBC2，其中最重要的為PBC1的數(shù)值

ENCODE對(duì)文庫(kù)復(fù)雜度的要求如下，

PBC1	PBC2	Bottlenecking level	NRF	Complexity	Flag colors
< 0.7	<1	Severe	< 0.7	Concerning	Orange
0.7 <= PBC1 <= 0.9	1 <= PBC2 <= 3	Moderate	0.7 <= NRF <= 0.9	Acceptable	Yellow
> 0.9	>3	None	> 0.9	ldeal	None

Replication quality metrics (重復(fù)性質(zhì)量檢測(cè))

IDR (Irreproducible Discovery Rate) plots (不可重復(fù)發(fā)現(xiàn)率)
（可選，僅在有至少2組重復(fù)(rep1,rep2,...)時(shí)計(jì)算）

IDR主要比較兩個(gè)樣本之間的重復(fù)性，先對(duì)peaks進(jìn)行排序，然后進(jìn)行重復(fù)性的分析，得到IDR得分。IDR<0.05，認(rèn)為有重復(fù)性，黑色表示，IDR>=0.05認(rèn)為沒(méi)有重復(fù)性，紅色表示。

• 一般對(duì)兩重復(fù)樣本進(jìn)行IDR檢測(cè)（rep1 vs rep2），檢測(cè)兩樣本之間的重復(fù)性。

• 單樣本的虛擬樣本間也可進(jìn)行IDR檢測(cè)（rep1-pr1 vs rep1-pr2），驗(yàn)證IDR檢測(cè)的準(zhǔn)確性。

• 樣本混樣成pool進(jìn)行IDR檢測(cè)（pool-pr1 VS pool-pr2）

Reproducibility QC and peak detection statistics（重復(fù)性QC及peak統(tǒng)計(jì)分析）

Reproducibility QC

• N1: Replicate 1 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep1 reads)

• N2: Replicate 2 self-consistent peaks (comparing two pseudoreplicates generated by subsampling Rep2 reads)

• Ni: Replicate i self-consistent peaks (comparing two pseudoreplicates generated by subsampling RepX reads)

• Nt: True Replicate consistent peaks (comparing true replicates Rep1 vs Rep2)

• Np: Pooled-pseudoreplicate consistent peaks (comparing two pseudoreplicates generated by subsampling pooled reads from Rep1 and Rep2)

• Self-consistency Ratio: max(N1,N2) / min (N1,N2)

• Rescue Ratio: max(Np,Nt) / min (Np,Nt)

• Reproducibility Test: If Self-consistency Ratio >2 AND Rescue Ratio > 2, then 'Fail' else 'Pass'

說(shuō)明：

• N1: replicate1自我虛擬peak的一致性peak

• N2: replicate2自我虛擬peak的一致性peak

• Ni: replicatei自我虛擬peak的一致性peak

• Nt: 真實(shí)一致性的peak

• Np: 整體的數(shù)據(jù)（pool）自我虛擬peak的一致性peak

• N optimal: 虛擬peak的一致性peak

• N conservation: 真實(shí)peak的一致性peak

• Self-consistency Ratio:   max(N1,N2) / min (N1,N2)

• Rescue Ratio:    max(Np,Nt) / min (Np,Nt)

• Reproducibility Test: 如果Self-consistency Ratio >2 AND Rescue Ratio > 2, 則失?。‵ailed）,否則通過(guò)（pass）

ENCODE對(duì)以上指標(biāo)的檢測(cè)標(biāo)準(zhǔn)如下：

Self-consistency Ratio	Rescue Ratio	Resulting Data Status	Flag colors
Less than 2	Less than 2	Ideal	None
Less than 2	Greater than 2	Acceptable	Yellow
Greater than 2	Less than 2	Acceptable	Yellow
Greater than 2	Greater than 2	Concerning	Orange

• IDR 一致的peak 最好大于70,000 ，不少于50,000 為可接受

Number of raw peaks （原始peaks數(shù)）

           Top 500000 raw peaks from macs2 with p-val threshold 0.01

說(shuō)明：

• 原始peak數(shù)最好不少于150,000，不少于100,000為可接受

Peak calling statistics （peak calling統(tǒng)計(jì)）

Peak region size （peak長(zhǎng)度范圍）

說(shuō)明：

• 圖形橫坐標(biāo)為region size，縱坐標(biāo)為peak數(shù)。

• Rep1與rep2顯示的是原始peaks（去除duplicate及線粒體基因組）

• Idr_opt顯示的是經(jīng)過(guò)IDR優(yōu)化后的peaks

• Overlap_opt顯示經(jīng)過(guò)overlap優(yōu)化后的peaks

Enrichment / Signal-to-noise ratio

Strand cross-correlation measures (trimmed/filtered SE BAM) (鏈互相關(guān)檢測(cè))

Performed on subsampled (15000000) reads mapped from FASTQs that are trimmed to 50.            Such FASTQ trimming and subsampling reads are for cross-corrleation analysis only.            Untrimmed FASTQs are used for all the other analyses.

NOTE1: For SE datasets, reads from replicates are randomly subsampled to 15000000.
           NOTE2: For PE datasets, the first end (R1) of each read-pair is selected and trimmed to 50 the reads are then randomly subsampled to 15000000.

• Normalized strand cross-correlation coefficient (NSC) = col9 in outFile

• Relative strand cross-correlation coefficient (RSC) = col10 in outFile

• Estimated fragment length = col3 in outFile, take the top value

NSC與RSC的算法如下：

從flitered BAM文件中虛擬一個(gè)含有25,000,000個(gè)reads的副本，并對(duì)該副本進(jìn)行鏈互相關(guān)分析。鏈互相關(guān)性分析主要顯示+，-鏈reads的相似性。由于正負(fù)reads的peak中心有偏移，因此，結(jié)果中一般會(huì)出現(xiàn)兩個(gè)peak，一個(gè)是標(biāo)準(zhǔn)peak，一個(gè)是虛擬peak（ Phantom Peak）。如下圖所示

Jensen-Shannon distance (filtered/deduped BAM)(JS散度檢測(cè))

說(shuō)明：

• JS散度是檢測(cè)兩樣本之間的離散程度，也就是檢測(cè)兩樣本之間的相似性。

Peak enrichment

Fraction of reads in peaks (FRiP) (peaks占總reads數(shù)的比值)

FRiP for macs2 raw peaks

FRiP for overlap peaks

FRiP for IDR peaks

For macs2 raw peaks:

• repX: Peak from true replicate X

• repX-prY: Peak from Yth pseudoreplicates from replicate X

• pooled: Peak from pooled true replicates (pool of rep1, rep2, ...)

• pooled-pr1: Peak from 1st pooled pseudo replicate (pool of rep1-pr1, rep2-pr1, ...)

• pooled-pr2: Peak from 2nd pooled pseudo replicate (pool of rep1-pr2, rep2-pr2, ...)

           For overlap/IDR peaks:

• repX_vs_repY: Comparing two peaks from true replicates X and Y

• repX-pr1_vs_repX-pr2: Comparing two peaks from both pseudoreplicates from replicate X

• pooled-pr1_vs_pooled-pr2: Comparing two peaks from 1st and 2nd pooled pseudo replicates

說(shuō)明：

• FRiP指的是fraction of all mapped reads that fall into peak regions. 代表peak區(qū)域內(nèi)reads的比例，peak區(qū)域內(nèi)的reads是抗體富集的序列，其他區(qū)域的序列是背景噪聲。 只有mapping到基因組上的reads才會(huì)進(jìn)行后續(xù)分析，所以直接用peak區(qū)域reads數(shù)目除以所有mapping基因組的數(shù)目，就得到了FRiP Score。

• Encode團(tuán)隊(duì)發(fā)現(xiàn)FRip Score和peak的個(gè)數(shù)存在正相關(guān)關(guān)系。 對(duì)于不同的轉(zhuǎn)錄因子chip數(shù)據(jù)，peak的個(gè)數(shù)不同，總體的趨勢(shì)來(lái)看，peak的個(gè)數(shù)越多，F(xiàn)RiP Score的值越大。在Encode的chip數(shù)據(jù)集中，有80%左右的FRiP Score值都超過(guò)了1%，所以將1%看做是一個(gè)衡量chip實(shí)驗(yàn)的軟標(biāo)準(zhǔn)。 之所以稱之為軟標(biāo)準(zhǔn)，是因?yàn)镕RiP score低于1%并不能直接說(shuō)明chip實(shí)驗(yàn)是失敗的，高于1%也不能直接說(shuō)明chip實(shí)驗(yàn)就是成功的。在ZNF274和RNA III型聚合酶的chip實(shí)驗(yàn)中，peak的個(gè)數(shù)很少，F(xiàn)RiP score的值也小于1%；在CTCF等轉(zhuǎn)錄因子實(shí)驗(yàn)中，F(xiàn)RiP score的值又遠(yuǎn)大于1%。

• 所以說(shuō)FRiP score值是和特定的轉(zhuǎn)錄因子或者組蛋白修飾相關(guān)，在研究特定轉(zhuǎn)錄因子時(shí)，參考別人已經(jīng)發(fā)表的數(shù)據(jù)中的FRiP Score作為衡量標(biāo)準(zhǔn)是更好的，在沒(méi)有參考的情況下，可以用1%的閾值來(lái)作為一個(gè)軟標(biāo)準(zhǔn)，為chip實(shí)驗(yàn)的質(zhì)量提供一個(gè)參考。當(dāng)peak個(gè)數(shù)的大于1000時(shí)，1%這個(gè)標(biāo)準(zhǔn)的效果還是比較好的，有相當(dāng)大的參考意義。